Processive Antitermination of Antibiotic Synthesis Genes

Abstract NusG is a transcription elongation protein used by virtually all organisms from the three domains of life. Bacterial NusG associates with RNA polymerase (RNAP) through its N-terminal domain, whilst, despite its small size, the C-terminal domain (CTD) forms dynamical interactions with other transcription factors (Rho, S10, NusB and NusA) to affect transcription elongation. While all bacteria encode for a core NusG, many also synthesize paralogs that transiently bind RNAP to alter expression of targeted genes. Yet, despite the importance of the genes they regulate, most of the known subfamilies of NusG paralogs have not been investigated in depth (e.g., UpxY, TaA, and ActX). We recently discovered a new and widespread subfamily of NusG-like proteins, which we called LoaP. Our preliminary investigation of this unique protein showed that Bacillus velezensis LoaP activates expression of a regulon that is comprised of two different antibiotic synthesis operons. Upon further inspection, we found evidence that suggests a broad regulatory relationship between LoaP and antibiotic synthesis operons. This discovery is particularly important because our data suggests that the LoaP regulatory protein reconfigures the transcription elongation machinery into an antitermination complex, capable of bypassing multiple termination sites spread throughout the antibiotic synthesis operons. The presence of these termination sites suggests that these operons have become ‘addicted’ to the LoaP antitermination factor, as their transcription would be impossible without the dedicated antitermination complex. We speculate that this observation explains why some antibiotic synthesis operons do not express well within the confines of a heterologous host; they may have simply accrued termination sites that strikingly inhibit transcription elongation in the absence of their cognate antitermination factor. Together, these data demonstrate how there is an urgent need to better understand the genetic regulatory mechanisms affecting antibiotic synthesis operons, as this information will influence the strategies used for discovering novel antibiotics and will lead to new tools for improving heterologous production of antibiotics. Therefore, it is of significant importance to understand how the LoaP antitermination mechanism exerts its regulatory influence over these important specialized metabolite operons. Moreover, our preliminary data have demonstrated that LoaP uses a regulatory mechanism that is different than those utilized by the other known NusG paralogs. In this project we will discover the molecular mechanism used by LoaP to specifically manipulate transcription elongation of antibiotic synthesis operons. This will significantly expand knowledge of the regulatory mechanisms that control antibiotic synthesis while also revealing new and fundamental insight into the workings of the transcription elongation complex.